Neurotransmission relies on the proper trafficking of synaptic vesicles in nerve terminals. Recently, a series of genetic studies have unmasked a fundamental role for phosphoinositides (i.e.phosphorylated derivatives of phosphatidylinositol, Ptdlns) and other lipids in this process. In particular, ablation of the two main enzymes regulating the levels of PIP2 at the synapse, PIPK1 gamma and the phosphoinositide phosphatase synaptojanin 1, was shown to produce defects at multiple stages in the synaptic vesicle cycle, thereby reflecting the multifaceted role of PIP2 in signaling at the synapse. Although a role for PIP2 in membrane traffic is now well established, several fundamental questions have emerged concerning how distinct pools of this lipid may exert specialized functions in such compartments as the plasma membrane. First, what are the molecular mechanisms ensuring the local regulation of PIP2 synthesis? Based on growing evidence showing that the small monomeric GTPase Arf6 is a major regulator of PIPK1 gamma and vesicular transport to and from the plasma membrane, we will explore its potential role in synaptic vesicle trafficking. Second, what are the mechanisms controlling the spatial restriction of PIP2 dephosphorylation at the membrane? An interesting hypothesis is that the PIP2 hydrolysis machinery may utilize membrane curvature sensors, such as BAR proteins, to eliminate this lipid preferentially from curved (i.e.in the endocytic bud),rather than flat, membranes. This hypothesis is strongly supported by studies showing the tight partnership between the membrane curvature sensor, endophilin, and synaptojanin 1, as well as by our preliminary biochemical data. Third, what are the main effectors for PIP2 at the synapse? In addition to components of the exo-endocytic machinery, major phospholipid-metabolizing enzymes, such as phospholipase D (PLD), have a strong requirement for PIP2 as a cofactor for their function. Since PLD enzymes are primary sources for the signaling lipid, phosphatidic acid, the metabolism of PIP2 is tightly linked to that of this phospholipid. Therefore, we will investigate the significance of this crosstalk as well as the role of PLD-derived phosphatidic acid for membrane traffic at the synapse. To test our hypotheses, we will utilize mouse genetics as well as lentivirus-mediated delivery of short hairpin RNAs and dominant interfering DNA constructs into cultured neurons. Effects of these manipulations will be analyzed using biochemical, ultrastructural and live fluorescence imaging approaches. Altogether, we anticipate that our research will significantly advance our understanding of how phospholipids regulate membrane traffic at the synapse.